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Ex vivo 31P NMR – A tool for phosphorus profiling in microbial cells – Towards phosphoromics
S206
Abstracts / New Biotechnology 33S (2016) S1–S213
P36-4 Biosynthesis of diastereomeric mannosylerythritol lipid using sugar-transferase from Pseudozyma tsukubaensis Azusa Saika 1,∗ , Hideaki Koike 1 , Tokuma Fukuoka 1 , Shuhei Yamamoto 2 , Takahide Kishimoto 2 , Tomotake Morita 2 1 National Institute of Advanced Industrial Science and Technology, Japan 2 Toyobo Co., Ltd., Japan
Mannosylerythritol lipids (MELs) are glycolipid biosurfactants, and produced by various fungi. While the conventional MELs composed of 4-O--d-mannopyranosyl-(2S,3R)-erythritol are produced by various fungal producers, a basidiomycetous yeast Pseudozyma tsukubaensis produces MEL-B, which contains 4-O--d-mannopyranosyl-(2R,3S)-erythritol as the sugar moiety (diastereomeric MEL-B) in large amount. Compared with the conventional MELs, genetic basis for the diastereomeric MEL biosynthesis in P. tsukubaensis is still unknown. Here, we identified a gene cluster involved in the MEL biosynthesis in P. tsukubaensis. A gene PtEMT1, which encodes erythritol/mannose transferase and plays a crucial role in determining chirality of the sugar moiety, shared high identity (69%) with homologs from strains in the genera Ustilago, Melanopsichium, Sporisorium and Pseudozyma. On phylogenetic analysis, PtEMT1p is categorized into an independent clade from the other ones. To biosynthesize diastereomeric MEL-A in the conventional MEL producer, we introduced the gene into a P. antarctica mutant strain, PaEMT1, which lacks MEL biosynthesis ability. From TLC analysis, the gene PtEMT1 from P. tsukubaensis NBRC1940 restored MEL production in PaEMT1. Based on NMR analysis, we succeeded for the first time that biosynthesis the diastereomeric MEL-A by gene engineering methods. This study will facilitate the expansion of structural and functional varieties of MELs. This work has been supported by Japan Science and technology Agency (JST) under Adaptable and Seamless Technology Transfer Program through Target-driven R&D. http://dx.doi.org/10.1016/j.nbt.2016.06.1430
P36-5 The possibilities to use Galactomyces geotrichum mould to enrich dairy products in functional compounds Anna Grygier ∗ , Kamila Myszka, Magdalena Rudzinska ´ Poznan University of Life Sciences, Poland Functional foods are increasingly choose by the consumers. The goal of their consumption is a positive effect on the human health. An interesting matrix for that products can be a dairy products which are popular in each age group, from children to elderly. In some traditional dairy products like fried cottage cheese, bryndza cheese, raclette cheese, kumys, shubat or nunu an interesting mould – Galactomyces geotrichum is present. This microorganism is a filamentous fungi, which is important to obtain characteristically taste and aroma of some dairy products. It is able to utilize lactic acid, involve hydrolysis of triglycerides and proteins. Otherwise Galactomyces geotrichum mould products peptides in kumys, that could inhibit angiotensin I converting enzyme. This peptides are used for the regulation of blood pressure in humans (Chaves-Lopez et al., 2012). The new research shows that the Galactomyces geotrichum mould isolated from the fried cottage cheese is able to production polyunsaturated fatty acids. These acids are important for the
prevention of cardiovascular diseases, when are eaten in correct proportions. Identification and quantification was prepared by GCFID method. Vitamin B6 and trehalose were detected by HPLC method. Vitamin B6 is involved in many aspects of macronutrient metabolism, neurotransmitter synthesis, histamine synthesis, hemoglobin synthesis and function, and gene expression. Trehalose is a natural disaccharide and is an equivalent to glucose. Trehalose has about 45% the sweetness of sucrose at concentrations above 22%. All of this components synthetized by Galactomyces geotrichum mould can be use for the functional dairy products. http://dx.doi.org/10.1016/j.nbt.2016.06.1431
P36-6 Ex vivo 31P NMR – A tool for phosphorus profiling in microbial cells – Towards phosphoromics Dorota Wieczorek ∗ , Jacek Lipok Opole University, Poland Phosphorus is an essential element in every living organisms. In the cells, it can be found mainly in the form of phosphate groups, which are part of various molecules such as: nucleic acids, derivatives of nucleotide, proteins, fat or carbohydrates. These organophosphorus substances play crucial role in cells’ development and functioning, therefore they may be good representatives of the metabolic condition of cell. Phosphorus nuclear magnetic resonance (31 P NMR technique) is the method, which allow to study the chemical nature of compounds containing phosphorus atom(s) in their structure. 31 P NMR is the routine NMR technique which is currently used for characterization of chemical structures of phosphorus compounds, and rarely for their quantitative analysis. However, relatively high sensitivity of magnetically influenced nuclei of phosphorus enables to differentiate phosphorus compounds, even if their chemical structures are similar. Such an attempt opens the way to metabolomic studies – mainly monitoring of energy status of cells or screening of different forms of phosphorus in various “living matrices”. The utility of 31 P NMR techniques in determination of various phosphorus compounds occurring in the cells of filamentous fungi was studied. The results of NMR analysis of extracts obtained from different strains of molds prove that phosphorus nuclear magnetic resonance is useful and relatively simple way for determination of cells phosphorus profile. http://dx.doi.org/10.1016/j.nbt.2016.06.1432
P36-7 Investigation on the fermentation for high adenosine and N6(2-hydroxyethyl)-adenosine (HEA) productions of Cordyceps cicadae Bo-Jun Ke ∗ , Chun-Lin Lee National Taitung University, Taiwan Cordyceps cicadae is a well-known Chinese herbal medicine for treating palpitations and eye diseases. The wild Cordyceps cicadae grows in high humidity conditions on mountains between 80 and 500 m high in Asia. It contains large quantities of adenosine, N6 -(2-hydroxyethyl)-adenosine (HEA), and other active constitutions, which are reported to have effects on anti-oxidation,
Abstracts / New Biotechnology 33S (2016) S1–S213
P36-4 Biosynthesis of diastereomeric mannosylerythritol lipid using sugar-transferase from Pseudozyma tsukubaensis Azusa Saika 1,∗ , Hideaki Koike 1 , Tokuma Fukuoka 1 , Shuhei Yamamoto 2 , Takahide Kishimoto 2 , Tomotake Morita 2 1 National Institute of Advanced Industrial Science and Technology, Japan 2 Toyobo Co., Ltd., Japan
Mannosylerythritol lipids (MELs) are glycolipid biosurfactants, and produced by various fungi. While the conventional MELs composed of 4-O--d-mannopyranosyl-(2S,3R)-erythritol are produced by various fungal producers, a basidiomycetous yeast Pseudozyma tsukubaensis produces MEL-B, which contains 4-O--d-mannopyranosyl-(2R,3S)-erythritol as the sugar moiety (diastereomeric MEL-B) in large amount. Compared with the conventional MELs, genetic basis for the diastereomeric MEL biosynthesis in P. tsukubaensis is still unknown. Here, we identified a gene cluster involved in the MEL biosynthesis in P. tsukubaensis. A gene PtEMT1, which encodes erythritol/mannose transferase and plays a crucial role in determining chirality of the sugar moiety, shared high identity (69%) with homologs from strains in the genera Ustilago, Melanopsichium, Sporisorium and Pseudozyma. On phylogenetic analysis, PtEMT1p is categorized into an independent clade from the other ones. To biosynthesize diastereomeric MEL-A in the conventional MEL producer, we introduced the gene into a P. antarctica mutant strain, PaEMT1, which lacks MEL biosynthesis ability. From TLC analysis, the gene PtEMT1 from P. tsukubaensis NBRC1940 restored MEL production in PaEMT1. Based on NMR analysis, we succeeded for the first time that biosynthesis the diastereomeric MEL-A by gene engineering methods. This study will facilitate the expansion of structural and functional varieties of MELs. This work has been supported by Japan Science and technology Agency (JST) under Adaptable and Seamless Technology Transfer Program through Target-driven R&D. http://dx.doi.org/10.1016/j.nbt.2016.06.1430
P36-5 The possibilities to use Galactomyces geotrichum mould to enrich dairy products in functional compounds Anna Grygier ∗ , Kamila Myszka, Magdalena Rudzinska ´ Poznan University of Life Sciences, Poland Functional foods are increasingly choose by the consumers. The goal of their consumption is a positive effect on the human health. An interesting matrix for that products can be a dairy products which are popular in each age group, from children to elderly. In some traditional dairy products like fried cottage cheese, bryndza cheese, raclette cheese, kumys, shubat or nunu an interesting mould – Galactomyces geotrichum is present. This microorganism is a filamentous fungi, which is important to obtain characteristically taste and aroma of some dairy products. It is able to utilize lactic acid, involve hydrolysis of triglycerides and proteins. Otherwise Galactomyces geotrichum mould products peptides in kumys, that could inhibit angiotensin I converting enzyme. This peptides are used for the regulation of blood pressure in humans (Chaves-Lopez et al., 2012). The new research shows that the Galactomyces geotrichum mould isolated from the fried cottage cheese is able to production polyunsaturated fatty acids. These acids are important for the
prevention of cardiovascular diseases, when are eaten in correct proportions. Identification and quantification was prepared by GCFID method. Vitamin B6 and trehalose were detected by HPLC method. Vitamin B6 is involved in many aspects of macronutrient metabolism, neurotransmitter synthesis, histamine synthesis, hemoglobin synthesis and function, and gene expression. Trehalose is a natural disaccharide and is an equivalent to glucose. Trehalose has about 45% the sweetness of sucrose at concentrations above 22%. All of this components synthetized by Galactomyces geotrichum mould can be use for the functional dairy products. http://dx.doi.org/10.1016/j.nbt.2016.06.1431
P36-6 Ex vivo 31P NMR – A tool for phosphorus profiling in microbial cells – Towards phosphoromics Dorota Wieczorek ∗ , Jacek Lipok Opole University, Poland Phosphorus is an essential element in every living organisms. In the cells, it can be found mainly in the form of phosphate groups, which are part of various molecules such as: nucleic acids, derivatives of nucleotide, proteins, fat or carbohydrates. These organophosphorus substances play crucial role in cells’ development and functioning, therefore they may be good representatives of the metabolic condition of cell. Phosphorus nuclear magnetic resonance (31 P NMR technique) is the method, which allow to study the chemical nature of compounds containing phosphorus atom(s) in their structure. 31 P NMR is the routine NMR technique which is currently used for characterization of chemical structures of phosphorus compounds, and rarely for their quantitative analysis. However, relatively high sensitivity of magnetically influenced nuclei of phosphorus enables to differentiate phosphorus compounds, even if their chemical structures are similar. Such an attempt opens the way to metabolomic studies – mainly monitoring of energy status of cells or screening of different forms of phosphorus in various “living matrices”. The utility of 31 P NMR techniques in determination of various phosphorus compounds occurring in the cells of filamentous fungi was studied. The results of NMR analysis of extracts obtained from different strains of molds prove that phosphorus nuclear magnetic resonance is useful and relatively simple way for determination of cells phosphorus profile. http://dx.doi.org/10.1016/j.nbt.2016.06.1432
P36-7 Investigation on the fermentation for high adenosine and N6(2-hydroxyethyl)-adenosine (HEA) productions of Cordyceps cicadae Bo-Jun Ke ∗ , Chun-Lin Lee National Taitung University, Taiwan Cordyceps cicadae is a well-known Chinese herbal medicine for treating palpitations and eye diseases. The wild Cordyceps cicadae grows in high humidity conditions on mountains between 80 and 500 m high in Asia. It contains large quantities of adenosine, N6 -(2-hydroxyethyl)-adenosine (HEA), and other active constitutions, which are reported to have effects on anti-oxidation,